Virtual microscope system

ABSTRACT

A virtual microscope system capable of obtaining a stained sample image and a statistical data of spectra in a short period of time is provided, the virtual microscope system includes an image obtaining unit for obtaining a stained sample image, a spectrum obtaining unit for obtaining a spectrum of the stained sample image, an optical path setting unit for setting an optical path of a light flux passed through the stained sample with respect to the image obtaining unit and the spectrum obtaining unit and a control unit for controlling to repeat obtaining the stained sample image by the image obtaining unit and obtaining the spectrum of the stained sample image by the spectrum obtaining unit in the observation field of the stained sample to create a virtual slide and a spectrum table of the stained sample.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority from Japanese Application No.2009-256312 filed on Nov. 9, 2009, the content of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to virtual microscope systems forestimating spectra of a stained sample image obtained by capturing astained sample.

BACKGROUND OF THE INVENTION

The spectral transmittance spectrum is one of physical quantitiesrepresenting a physical property specific to a subject of imaging. Thespectral transmittance is a physical quantity representing a ratio oftransmitted light to incident light at each wavelength and, unlike thecolor information such as an RGB value that depends on the change ofillumination light, is the information specific to an object, whosevalues do not change depending on extrinsic influences. Thus thespectral transmittance is used in various fields as the information toreproduce the color of the subject itself. For example, in the field ofpathology diagnosis that uses body tissue samples, particularly,pathological specimens, the spectral transmittance has been used, as anexample of a spectral characteristic value, for analyzing imagesobtained by capturing samples. The examples of use of the spectraltransmittance for pathology diagnosis will now be described below indetail.

As one of the pathological examinations for pathology diagnosis, thetissue diagnosis by which tissue is taken from a lesioned part and isobserved with a microscope for diagnosing a disease or the degree oflesion expansion is known. This tissue diagnosis is also called“biopsy”, by which a block sample obtained by organ harvesting or apathological specimen obtained by needle biopsy is sliced into severalmicrometers thick and is magnified with a microscope to obtain variousfindings, and this diagnosis has been widely used. Transmissionobservation using an optical microscope is one of the most commonobservation methods, because equipment and material are relativelyinexpensive and easy to be handled, and the method has been used formany years. In the case of the transmission observation, the slicedsamples absorb and scatter almost no light and are almost transparentand colorless. Thus, the samples are generally stained by a dye prior toobservation.

Various staining methods have been proposed, and more than a hundredmethods have been proposed in total. Particularly, for pathologicalspecimens, hematoxylin-eosin staining (hereinafter referred to as “HEstaining”) that uses bluish-purple hematoxylin and red eosin as pigmenthas been used as a standard staining method.

Hematoxylin is a natural substance extracted from plants, and has nostainability itself. However, hematin, an oxidative product ofhematoxylin, is a basophilic dye and binds to a negatively chargedsubstance. Deoxyribonucleic-acid (DNA) contained in cell nucleus isnegatively charged by a phosphate group contained as a component and,thus is stained bluish-purple when it binds to hematin. As describedabove, a substance having stainability is its oxidative product,hematin, and is not hematoxylin. However, hematoxylin is commonly usedas a name of dye, and this applies to the following explanations.

On the other hand, eosin is an acidophilic dye and binds to a positivelycharged substance. Amino acid and protein are charged negatively orpositively depending on its pH environment, and have a strong tendencyto be charged positively under acidity. Thus, there are some cases whereacetic acid is added to eosin solution. The protein contained incytoplasm is stained red or pale red when binding to eosin.

After being HE stained (a stained sample), cell nucleuses, bone tissuesand the like are stained bluish-purple, and cytoplasm, connectivetissue, blood cells and the like are stained red in the sample, whichoffers visibility. Thus an observer understands the size and positionalrelation and the like of elements constituting tissues such as cellnucleuses, thereby enabling the observer to determine a state of thesample morphologically.

In addition to a visual inspection by the observer, the stained samplecan also be observed by multiband imaging displayed on a display screenof an external device. In the case where images are displayed on ascreen, processing for estimating the spectral transmittance at eachpoint of the sample from the obtained multiband image and processing forestimating dye amount of a dye that stains the sample based on theestimated spectral transmittance and the like are performed and, theimage to be displayed, which is an RGB image of the sample for display,is composed.

Methods of estimating the spectral transmittance at each point of thesample from the multiband image of the sample include, for example,principal component analysis, Wiener estimation method and the like. TheWiener estimation is widely known as one of the linear filtering methodsfor estimating an original signal from an observed signal on which noiseis superimposed and is a method for minimizing errors in view of thestatistical properties of the observed object and the noise (observednoise). Signals from a camera contain some sort of noise. Thus theWiener estimation is an extremely useful method for estimating anoriginal signal.

A method of creating a virtual slide by composing a display image from amultiband image of a sample is described below. The virtual slide is animage created by patching one or more multiband images captured by amicroscope device and, for example, an image created by patching aplurality of high-resolution images at each portion of a stained samplecaptured by a high-power microscope objective lens. The virtual slidemeans a wide-field and high-definition multiband image of the entireview of a stained sample.

First, a multiband image of a sample is captured, for example, based ona frame sequential method, while rotating a filter wheel to switch 16pieces of bandpass filters. In this manner, multiband images having apixel value of 16 bands can be obtained at each point of the sample.Although the dye is originally distributed three-dimensionally in asample to be observed, the dye cannot be captured as a three-dimensionalimage as it is by using an ordinary transmission observation system, andthe illumination light that has passed the sample is observed as atwo-dimensional image projected on an imaging element of a camera.Therefore, each point described herein means a point at the samplecorresponding to each pixel of the projected imaging element.

For an arbitrary point (pixel) x of a captured multiband image, arelation expressed by the following Equation (1) based on a responsesystem of a camera is established between the pixel value g(x, b) in theband b and the spectral transmittance t(x, λ) at the corresponding pointof the sample.

g(x,b)=∫_(λ)ƒ(b,λ)s(λ)e(λ)t(x,λ)dλ+n(b)  (1)

In the Equation (1), denotes wavelength, f(b,λ) denotes spectraltransmittance of a “b”th filter, s(λ) denotes spectral sensitivitycharacteristic of a camera, e(λ) denotes spectral emissioncharacteristic of illumination, and n(b) denotes observation noise inthe band b. b denotes a serial number for identifying the band, and isan integer satisfying 1≦b≦16. In the practical calculation, thefollowing Equation (2) obtained by discretizing the Equation (1) in awavelength direction is used.

G(x)=FSET(x)+N  (2)

In the Equation (2), when the number of sample points in a wavelengthdirection is designated as D and the number of bands is designated as B(in this case, B=16), G(x) denotes a matrix of B rows by one columncorresponding to the pixel value g(x, b) at the point x. In the samemanner, T(x) denotes a matrix of D rows by one column corresponding tot(x, λ), and F denotes a matrix of B rows by D columns corresponding tof(b, λ). On the other hand, S denotes a diagonal matrix of D rows by Dcolumns, and a diagonal element corresponds to s(λ). In the same manner,E denotes a diagonal matrix of D rows by D columns, and a diagonalelement corresponds to e(λ). N denotes a matrix of B rows by one columncorresponding to n(b). In the Equation (2), because equations of aplurality of bands are put together using a matrix, a variable brepresenting a band is not described. Further, an integral of thewavelength λ is replaced by a product of matrices.

To simplify the description, the matrix H defined by the followingEquation (3) is introduced. The matrix H is also called a system matrix.

H=FSE  (3)

Thus, the Equation (2) is replaced by the following Equation (4).

G(x)=HT(x)+N  (4)

Next, the spectral transmittance at each point of the sample isestimated from the captured multiband image by using the Wienerestimation. The estimation value of the spectral transmittance (spectraltransmittance data), T̂(x), can be calculated by the following Equation(5). T̂ indicates that a symbol, “̂(hat)”, representing an estimationvalue, is put over T.

{circumflex over (T)}(x)=(x)  (5)

W is expressed by the following Equation (6), and is referred to as“Wiener estimation matrix” or “estimation operator used for the Wienerestimation”.

W=R _(SS) H ^(t)(R _(SS) H ^(t) +R _(NN))⁻¹  (6)

Where, ( )^(t): transposed matrix, and ( )⁻¹: inverse matrix.

In the Equation (6), R_(SS) is a matrix of D rows by D columns andrepresents an autocorrelation matrix of spectral transmittance of thesample, and R_(NN) is a matrix of B rows by B columns and represents anautocorrelation matrix of noise of the camera used for capturing.

In order to calculate an estimation operator W by which each mainelement such as cell nucleus, cytoplasm, blood cell, cavum and the likecan appropriately be estimated, spectrum of each main element such ascell nucleus, cytoplasm, blood cell, cavum and the like are neededpreviously. Thus the user needs to measure the spectrum of each mainelement of the sample previously with a spectrometer while moving themeasuring position, which may be troublesome.

In order to solve the above mentioned problem, for example, the JapaneseUnexamined Patent Application Publication No. 2009-014354 discloses animage processing device by which an appropriate estimation operator, W,is calculated automatically. In this image processing device, a spectrumof each main element of a sample is measured with a spectrometer whilemoving the estimating position automatically, and an estimation operatorW is calculated from the measured spectrum. Then, the estimationoperator W is evaluated and if it is not appropriate, a spectrum of eachmain element of the sample is measured again and, thus an appropriateestimation operator W is calculated automatically.

According to the image processing device disclosed in the aforementioneddocument, an appropriate estimation operator W is calculatedautomatically, thereby reducing a burden on the user. However, theinventor of the present invention considers that, in the aforementionedimage processing device, spectrum of a plurality of elements of a sampleshould be measured previously with a spectrometer while moving themeasurement position. Thus the processing requires a lot of time and asa result, estimation of spectrum of the sample will be time-consuming.

In view of the aforementioned problem, the object of the presentinvention is to provide a virtual microscope system by which a stainedsample image obtained by capturing the stained sample and statisticaldata of spectra can be obtained in a short period of time.

SUMMARY OF THE INVENTION

The first aspect of the invention which achieves the aforementionedobjects is a virtual microscope system for capturing a stained sample toestimate a spectrum, which includes:

an image obtaining unit for obtaining a stained sample image of one ormore bands of the stained sample;

a spectrum obtaining unit for obtaining a spectrum at one or morepredetermined portions of the stained sample image;

an optical path setting unit for setting an optical path of a light fluxpassed through the stained sample, with respect to the image obtainingunit and the spectrum obtaining unit, so that the spectrum of thestained sample image can be obtained by the spectrum obtaining unit eachtime the image obtaining unit obtains the stained sample image; and

a control unit for controlling, in two or more observation fields of thestained sample, to repeat obtaining the stained sample image by theimage obtaining unit and obtaining the spectrum of the stained sampleimage by the spectrum obtaining unit so that a virtual slide and aspectrum table of the stained sample are created.

The second aspect of the invention resides in the virtual microscopesystem in accordance with the first aspect, wherein

the optical path setting unit has an optical path switching mirror forswitching the optical path of the light flux so that the light fluxpassed through the stained sample is selectively incident on the imageobtaining unit or the spectrum obtaining unit.

The third aspect of the invention resides in the virtual microscopesystem in accordance with the first aspect, wherein

the optical path setting unit has a disposition switching mechanism forselectively placing the image obtaining unit or the spectrum obtainingunit on the optical path of the light flux passed through the stainedsample.

The fourth aspect of the invention resides in the virtual microscopesystem in accordance with the first aspect, wherein

the optical path setting unit has a beam splitter for splitting theoptical path of the light flux so that the light flux passed through thestained sample is incident on the image obtaining unit and the spectrumobtaining unit simultaneously.

The fifth aspect of the invention resides in the virtual microscopesystem in accordance with the first aspect, wherein

the image obtaining unit is any one of an RGB camera, a monochromecamera, a two or more bands camera, and a multiband camera provided witha camera and an optical filter.

The sixth aspect of the invention resides in the virtual microscopesystem in accordance with the first aspect, wherein

the spectrum obtaining unit has an optical magnification increasing unitfor magnifying the stained sample image and obtains the spectrum fromthe stained sample image magnified by the optical magnificationincreasing unit.

The seventh aspect of the invention resides in the virtual microscopesystem in accordance with the first aspect, further including:

a spectrum obtaining position pixel value calculating unit for obtaininga pixel value at the position of the spectrum obtained by the spectrumobtaining unit from the stained sample image obtained by the imageobtaining unit, wherein

as the spectrum table, a spectrum table containing at least the spectrumand the pixel value is created.

The eighth aspect of the invention resides in the virtual microscopesystem in accordance with the first aspect, further including:

an estimation operator calculating unit for calculating an estimationoperator from the spectrum table; and

a spectrum estimating unit for estimating a spectrum of a pixel thatmakes up the virtual slide by using the estimation operator.

The ninth aspect of the invention resides in the virtual microscopesystem in accordance with the eighth aspect, further including:

a spectrum selecting unit for selecting a plurality of spectracorresponding to the pixel value of the pixel that makes up the virtualslide from the spectrum table, wherein

the estimation operator calculating unit calculates an estimationoperator for each pixel value from the plurality of spectra selected bythe spectrum selecting unit; and

the spectrum estimating unit estimates the spectrum of the pixel thatmakes up the virtual slide by using the estimation operator for each ofthe pixel value.

The tenth aspect of the invention resides in the virtual microscopesystem in accordance with the eighth aspect, further including:

a spectrum selecting unit for selecting a spectra corresponding to thepixel value of the pixel that makes up the virtual slide from thespectrum table, wherein

the spectrum selected by the spectrum selecting unit is a spectrumestimation value.

The eleventh aspect of the invention resides in the virtual microscopesystem in accordance with the seventh aspect, wherein

the pixel value stored in the spectrum table is any one of an obtainedpixel value, a pixel value converted to a color space and a featurevalue calculated from the pixel value.

The twelfth aspect of the invention resides in the virtual microscopesystem in accordance with the seventh aspect, wherein

the spectrum table consists of a data set containing at least thespectrum, the pixel value and the information of the pixel position fromwhich the spectrum is obtained.

The thirteenth aspect of the invention resides in the virtual microscopesystem in accordance with the seventh aspect, wherein

the spectrum obtaining position pixel value calculating unit calculatesany one of a pixel value of the center pixel in an obtaining area of thespectrum, a statistical value of the pixel value of the pixel in theobtaining area, and a value calculated by convolving the pixel value inthe obtaining area with a light-receiving characteristic of the spectrumobtaining unit.

The virtual microscope system according to the present invention cancreate statistical data of spectra and a virtual slide almostsimultaneously. Thus a spectra of a stained sample can be estimated athigh speed.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram for illustrating the principles of a virtualmicroscope system in accordance with a first embodiment of the presentinvention;

FIG. 2 is a diagram for illustrating the principles of the virtualmicroscope system in accordance with the first embodiment of the presentinvention;

FIG. 3 is a diagram showing a modification example of a spectrumobtaining unit shown in FIG. 1;

FIG. 4 is a diagram showing another configuration of an optical pathsetting unit shown in FIG. 1;

FIG. 5 is a functional block diagram showing a configuration of thevirtual microscope system in accordance with the first embodiment;

FIG. 6 is a block diagram showing a configuration of main parts of animage obtaining unit shown in FIG. 5;

FIG. 7 is a diagram showing a spectral sensitivity characteristic of anRGB camera shown in FIG. 6;

FIG. 8 is a diagram showing a spectral transmittance characteristic oftwo optical filters constituting a filter unit shown in FIG. 6;

FIG. 9 is a diagram showing a specific configuration of a microscopeapparatus shown in FIG. 5;

FIG. 10 is a flow chart showing a creation process of a virtual slideand a spectrum table by the virtual microscope system shown in FIG. 5;

FIG. 11 is a flow chart showing an estimation process of the spectrum bythe virtual microscope system shown in FIG. 5;

FIG. 12 is a diagram for illustrating the principles of a virtualmicroscope system in accordance with a second embodiment of the presentinvention;

FIG. 13 is a functional block diagram showing a configuration of thevirtual microscope system in accordance with the second embodiment;

FIG. 14 is a flow chart showing a creation process of a virtual slideand a spectrum table by the virtual microscope system shown in FIG. 13;

FIG. 15 is a diagram showing an example of a spectrum table created by aprocess shown in FIG. 14;

FIG. 16 is a flow chart showing a spectrum estimation process by thevirtual microscope system shown in FIG. 13;

FIG. 17 is a diagram showing an object sample having a plurality oftissues; and

FIG. 18 is a diagram showing other configuration of the optical pathsetting unit shown in FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preferred embodiments of the present invention will now be described indetail with reference to the accompanying drawings. It should be notedthat the present invention is not limited by the embodiments shownbelow, and the same numerical symbols are assigned to the same portionsin the drawings.

First Embodiment

FIGS. 1 and 2 are diagrams for illustrating the principles of a virtualmicroscope system in accordance with a first embodiment of the presentinvention. In the virtual microscope system in accordance with thepresent embodiment, a stained sample (hereinafter referred to as an“object sample”) 11 that is placed on a stage of a microscope apparatusis illuminated by a light source 12 through an illumination opticalsystem 13, and the light transmitted therethrough is incident on anoptical path setting unit 15 through an observation optical system 14comprising a microscope objective lens. The optical path setting unit 15is, for example, constituted by an optical path switching mirror 15 a.The optical path switching mirror 15 a is constituted, for example, by areflective mirror that is evacuatable relative to the optical path ofthe incident light. The optical path of the incident light from theobservation optical system 14 is directed to an image obtaining unit 16by evacuating the optical path switching mirror 15 a from the incidentlight path, and the optical path of the incident light from theobservation optical system 14 is directed to a spectrum obtaining unit17 by inserting the optical path switching mirror 15 a into the incidentlight path.

As shown in FIG. 2( a), an object sample image 21 of one or more bandsof the object sample 11 is obtained by the image obtaining unit 16 withthe optical path switching mirror 15 a evacuated from the incident lightpath, and the obtained object sample image 21 is stored in a storageunit, which is not shown, as a portion of a virtual slide 22.Thereafter, a spectrum 24 (see FIG. 2( b)) of one or more predeterminedportions 23 (see FIG. 1) of the object sample image 21 obtained by theimage obtaining unit 16 is obtained by the spectrum obtaining unit 17with the optical path switching mirror 15 a inserted into the incidentlight path, and each obtained spectrum 24 of predetermined portions 23is registered in a spectrum table 25 as shown in FIG. 2. FIG. 1 shows anexample of obtaining the spectrum 24 of each of two predeterminedportions 23 of the object sample image 21. Obtaining the object sampleimage 21 and obtaining its spectrum 24 are repeated while moving theobject sample 11, thereby creating a virtual slide 22 of m×n pieces anda spectrum table 25 almost simultaneously.

In the above mentioned explanation, after obtaining the object sampleimage 21, the spectrum 24 at its predetermined portion 23 is obtained.However, the order may be reversed. In addition, the spectrum obtainingunit 17 may be constituted to obtain not only the spectrum 24 at each oftwo predetermined portions 23 of the object sample image 21 but also thespectrum 24 at one predetermined portion 23 or as shown in FIG. 3, thespectrum 24 at each of three or more predetermined portions 23 bymagnifying the incident light that is outputted from the observationoptical system 14 and passes through the optical path switching mirror15 a with a magnifying optical system 27.

Further, as shown in FIG. 4, the optical path setting unit 15 may beconstituted to split the optical path of the incident light from theobservation optical system 14 by using a beam splitter 15 b such as ahalf mirror, etc., and to set an optical path for the image obtainingunit 16 and for the spectrum obtaining unit 17 respectively. In thiscase, obtaining the object sample image 21 by the image obtaining unit16 and obtaining the spectrum 24 of the object sample image 21 by thespectrum obtaining unit 17 can be performed simultaneously.

In addition, when the optical path setting unit 15 is constituted byusing the evacuatable optical path switching mirror 15 a as shown inFIGS. 1 and 3, the transmitted light of the object sample 11 can be usedefficiently, thereby decreasing the illumination light amount comparedto the example shown in FIG. 4. Further, as shown in FIG. 4, when theoptical path setting unit 15 is constituted by using the beam splitter15 b, the virtual slide 22 and the spectrum table 25 can be created in ashorter time compared to FIGS. 1 and 3.

FIG. 5 is a functional block diagram showing a configuration of thevirtual microscope system in accordance with the present embodimentbased on the aforementioned principles. The virtual microscope systemconsists of a microscope apparatus 100 and a host system 200 thatcontrols the microscope apparatus 100 and performs creation of a virtualslide, estimation of spectrum and the like. The microscope apparatus 100includes an image obtaining unit 110, a spectrum obtaining unit 130, anoptical path setting unit 150 and a stage 170.

The image obtaining unit 110 (an equivalent of the image obtaining unit16 in FIG. 1) obtains an object sample image of one or more bands (inthis case, six-band image) of an object sample captured by a microscopeand, for example, as shown in FIG. 6 which shows a typical configurationof main parts, includes an RGB camera 111 having an imaging element suchas CCD (Charge Coupled Devices) and CMOS (Complementary Metal OxideSemiconductor) and a filter unit 113 that limits the wavelength band ofthe image forming light to the predetermined range.

The RGB camera 111 has, as shown in FIG. 7, for example, a spectralsensitivity characteristic of each of R(red), G(green) and B(blue)bands. The filter unit 113 includes a rotatory filter switching unit 115to which two pieces of optical filters 117 a and 117 b having differentspectral transmittance characters are held so that the transparentwavelength region of each of R, G and B bands is split into two. FIG. 8(a) shows a spectral transmittance characteristic of the optical filter117 a and FIG. 8( b) shows a spectral transmittance characteristic ofanother optical filter 117 b.

First, for example, the optical filter 117 a is located on an opticalpath extending from a light source 119 (an equivalent of the lightsource 12 in FIG. 1) to the RGB camera 111 so that the light source 119illuminates the object sample 11 placed on the stage 170, and thetransmitted light is formed on the RGB camera 111 through an imageforming optical system 121 (an equivalent of the observation opticalsystem 14 in FIG. 1) and the optical filter 117 a. In this manner, afirst capturing is performed. Thereafter, the filter switching unit 115is rotated to locate the optical filter 117 b on the optical pathextending from the light source 119 to the RGB camera 111. Thus, in thesame manner, a second capturing is performed.

In this manner, three-band images different from each other are obtainedby the first capturing and the second capturing, thereby obtainingmultiband images of six bands in total. It should be noted that thenumber of optical filters provided at the filter unit 113 is not limitedto two, and three or more optical filters may be used to obtain imagesof more bands. The obtained object sample image of the object sample 11is stored as a part of a virtual slide in a storage unit 230 of a hostsystem 200, which will be described below. The image obtaining unit 110may obtain only RGB images using the RGB camera 111.

In FIG. 5, a spectrum obtaining unit 130 (an equivalent of the spectrumobtaining unit 17 in FIG. 1) obtains a spectrum of one or morepredetermined portions of the object sample image obtained by the imageobtaining unit 110, and is provided with a spectrometer that uses anoptical fiber and silicon detector corresponding to the predeterminedportion. Thus, if a plurality of predetermined portions are set, aplurality of spectrometers are provided.

The optical path setting unit 150 sets an optical path of a light fluxconverged by the microscope objective lens with respect to the imageobtaining unit 110 and the spectrum obtaining unit 130 so that thespectrum obtaining unit 130 can obtain a spectrum of the predeterminedportion of the object sample image each time the image obtaining unit110 obtains an object sample image and, as shown in FIG. 1 or FIG. 4, isconstituted by the optical path switching mirror 15 a or the beamsplitter 15 b.

The stage 170 is used for placing an object sample thereon and forpositioning the object sample relative to the microscope objective lens,and is constituted by, for example, an electrical stage that is movablein a two-dimensional direction in a plane perpendicular to an opticalaxis of the microscope objective lens.

On the other hand, the host system 200 includes a control unit 210, astorage unit 230, a calculating unit 250, an input unit 270 and adisplay unit 290.

The input unit 270 is realized by input apparatus such as, for example,a keyboard, a mouse, a touch panel and various kinds of switches and, inresponse to the operation inputs, outputs input signals to the controlunit 210.

The display unit 290 is realized by a display apparatus such as an LCD(Liquid Crystal Display), an EL (Electro Luminescence) display, a CRT(Cathode Ray Tube) display and displays various screens based on thedisplay signals inputted from the control unit 210.

The calculating unit 250 includes an estimation operator calculatingunit 255, a spectrum estimating unit 257 and a virtual slide creatingunit 259. The virtual slide creating unit 259 processes each of aplurality of object sample images obtained by multiband capturing of aportion of the object sample 11 by the microscope apparatus 100, therebycreating a virtual slide image.

The storage unit 230 is realized by various IC memories such as a ROMand RAM, like a flash memory capable of updating and storing, datastorage media such as a hard disk integrated or connected by datacommunication terminals and a CD-ROM, and a reader thereof and the like.The storage unit 230 stores an image processing program 231 forprocessing images obtained by the image obtaining unit 110, a virtualslide creating program 233 for creating a virtual slide, a createdvirtual slide 235, a spectrum table 237 (an equivalent of the spectrumtable 25 in FIG. 2) obtained by the spectrum obtaining unit 130, data tobe used during program running and the like.

The control unit 210 includes an image obtaining control unit 211 forcontrolling the operation of the image obtaining unit 110 to obtainimages of the object sample, a spectrum obtaining control unit 213 forcontrolling the operation of the spectrum obtaining unit 130 to obtainspectra of the object sample, an optical path setting control unit 215for controlling the operation of the optical path setting unit 150 toswitch the optical path and a stage control unit 217 for controlling thestage 170 to move the observation field. The control unit 210 givesinstructions or transfers data to each unit constituting the virtualmicroscope system based on the input signal inputted from the input unit270, the object sample image inputted from the image obtaining unit 110,the program or the data stored in the storage unit 230 and the like,thereby controlling overall operation. The control unit 210 is realizedby hardware such as a CPU. When the optical path setting unit 150 isconstituted by the beam splitter 15 b as shown in FIG. 4, the opticalpath setting control unit 215 is not needed.

Although FIG. 5 shows a functional configuration of the host system 200,the actual host system 200 can be realized by a publicly known hardwareconfiguration provided with a CPU, a video board, and a main storageunit such as a main memory (RAM) and an interface device for connectingan external storage unit such as a hard disk and various storage media,a communication device, external inputs and the like. Thus,general-purpose computers such as a workstation and a personal computercan be used as the host system 200.

FIG. 9 is a diagram showing a specific configuration of the microscopeapparatus 100 shown in FIG. 5. The microscope apparatus 100 includes thestage 170 on which the object sample 11 is placed, a microscope body 127having an approximately U shape viewed laterally, that supports thestage 170 and holds an objective lens 125 (an equivalent of theobservation optical system 14 in FIG. 1) through a revolver 123, a lightsource 119 provided rearward of the bottom of the microscope body 127and a lens tube 129 placed on top of the microscope body 127. Further, abinocular unit 131 for visually observing the sample image of the objectsample 11 and a TV camera 133 (an equivalent of the RGB camera 111 inFIG. 6) for capturing the sample image of the object sample 11 andobtaining the object sample image are attached to the lens tube 129. Itshould be noted that, as shown in FIG. 9, the optical axis direction ofthe objective lens 125 is defined as a Z direction and the planeperpendicular to the Z direction is defined as an XY plane.

The stage 170 is configured to be movable in X,Y and Z directions. Inother words, the stage 170 is movable, in the XY plane, by a motor 135and an XY drive control unit 137 that controls drive of the motor 135.The XY drive control unit 137 detects a predetermined origin position inthe XY plane of the stage 170 by an origin sensor for the XY position(not shown) and controls the driving amount of the motor 135 based onthe origin position, thereby moving the observation field of the objectsample 11.

Further, the stage 170 is movable in the Z direction by a motor 139 anda Z drive control unit 141 that controls the drive of the motor 139. TheZ drive control unit 141 detects a predetermined origin position of thestage 170 in the Z direction by an origin sensor for the Z position (notshown) and controls the driving amount of the motor 139 based on theorigin point, thereby moving the focus of the object sample 11 to anarbitrary Z position in a predetermined height range.

The revolver 123 is rotatably held relative to the microscope body 127,and disposes the objective lens 125 above the object sample 11. Theobjective lens 125 is attached replaceably with other objective lenshaving a different magnification (observation magnification) relative tothe revolver 123 and is inserted into the optical path of theobservation light in response to the rotation of the revolver 123 toallow the objective lens 125 used for observation of the object sample11 to be switched alternatively.

The microscope body 127 includes therein an illumination optical system(an equivalent of the illumination optical system 13 in FIG. 1) forperforming transmitted illumination of the object sample 11 at thebottom thereof. In the illumination optical system, a collector lens 143for collecting illumination light outputted from the light source 119,an illumination system filter unit 145, a field stop 147, an aperturestop 149, a folding mirror 151 for deflecting an optical path of theillumination light along the optical axis of the objective lens 125, acondenser optical element unit 153, a top lens unit 155 and the like aredisposed along the optical path of the illumination light in place. Theobject sample 11 is subject to the illumination light outputted from thelight source 119 by the illumination optical system, and the transmittedlight therefrom is incident on the objective lens 125 as observationlight.

In addition, the microscope body 127 includes the spectrum obtainingunit 130 and the optical path setting unit 150 on an optical pathbetween the objective lens 125 and the lens tube 129 so that an opticalpath is set on the spectrum obtaining unit 130 and the lens tube 129.The optical path setting unit 150 is constituted by using the opticalpath switching mirror 15 a or the beam splitter 15 b shown in FIG. 1 orFIG. 4.

Further, the microscope body 127 includes therein a filter unit 157 onan optical path between the optical path setting unit 150 and the lenstube 129. The filter unit 157 is an equivalent of the filter unit 113shown in FIG. 7 and rotatably holds two or more optical filters 159 tolimit the wavelength band of the light that is focused as a sample imageto a predetermined range and appropriately inserts the optical filters159 into the optical path of the observation light behind the objectivelens 125.

The lens tube 129 includes therein a beam splitter 161 for switching theoptical path of the observation light passed through the filter unit 157to lead the light to the binocular unit 131 or the TV camera 133. Thesample image of the object sample 11 is introduced in the binocular unit131 by the beam splitter 161, and is observed visually by a microscopistthrough an eyepiece 163, or captured by the TV camera 133. The TV camera133 is constituted by including an imaging element such as CCD or CMOSthat forms a sample image (more specifically, a sample image in thefield range of the objective lens 125), captures the sample image andoutputs the image data of the sample image (object sample image) to thehost system 200.

The microscope apparatus 100 further includes a microscope controller165, a TV camera controller 167 and a spectrum obtaining controller 169.The microscope controller 165 controls overall operation of each partthat constitutes the microscope apparatus 100 under the control of thehost system 200. For example, the microscope controller 165 switches theobjective lens 125 disposed on the optical path of the observation lightby rotating the revolver 123 and makes adjustments of each part of themicroscope apparatus 100 associated with observation of the objectsample 11 such as dimming control of the light source 119 correspondingto the magnification, etc. of the switched objective lens 125, switchingof various optical elements or giving instructions for moving the stage170 to the XY drive control unit 137 and the Z drive control unit 141and the like, while the microscope controller 165 appropriately reportsthe state of each unit to the host system 200.

The TV camera controller 167 performs, under the control of the hostsystem 200, ON/OFF switching of the automatic gain control, setting ofgain, ON/OFF switching of the automatic exposure control, setting ofexposure time and the like to drive the TV camera 133 and controls thecapturing operation of the TV camera 133. In addition, the spectrumobtaining controller 169 controls, under the control of the host system200, the obtaining of a spectrum by the spectrum obtaining unit 130 andprovides the obtained spectrum to the host system 200.

Operation of the main parts of the virtual microscope system inaccordance with the present embodiment is now described below withreference to the flow charts shown in FIGS. 10 and 11.

FIG. 10 is a flow chart showing creation process of a virtual slide anda spectrum table. First, in the control unit 210, the stage control unit217 controls the operation of the stage 170 to move the observationfield of the object sample 11 (step S101). Next, the optical pathsetting control unit 215 controls the operation of the optical pathsetting unit 150 to direct an optical path to the image obtaining unit110 (step S103).

Thereafter, the image obtaining control unit 211 of the control unit 210controls the operation of the image obtaining unit 110 to obtain theobject sample image of the object sample 11 (step S105), and theobtained object sample image is stored in the storage unit 230 as a partof the virtual slide 235 (step S107).

Thereafter, the optical path setting control unit 215 of the controlunit 210 controls the operation of the optical path setting unit 150 todirect an optical path to the spectrum obtaining unit 130 (step S109).Then, the spectrum obtaining control unit 213 of the control unit 210controls the operation of the spectrum obtaining unit 130 to obtain thespectrum of the object sample 11 (step S111), and the obtained spectrumof the object sample 11 is stored in the spectrum table 237 of thestorage unit 230 (step S113). When the spectrum obtaining unit 130obtains spectrum of a plurality of portions of the object sample image,spectrum is obtained from all of the portions (step S115).

Thereafter, the control unit 210 repeats the steps from S101 to S115until the required field of the object sample 11 is obtained to createthe virtual slide 235 and the spectrum table 237 of the entire or a partof the object sample 11 (step S117). When the optical path setting unit150 is constituted by the beam splitter 15 b, as shown in FIG. 4, stepsS103 and S109 are not necessary. In this case, the obtaining process ofthe object sample image by the image obtaining unit 110 and theobtaining process of the spectrum by the spectrum obtaining unit 130 areperformed concurrently or in sequence.

In this manner, a spectrum of the object sample image is obtained by thespectrum obtaining unit 130 each time the object sample image isobtained by the image obtaining unit 110, thereby creating the virtualslide 235 and the spectrum table 237 at high speed. In particular, whenthe optical path setting unit 150 is constituted by the beam splitter 15b as shown in FIG. 4, time for switching the optical path switchingmirror 15 a, which is required when using the movable optical pathswitching mirror 15 a, is not needed, thus the virtual slide 235 and thespectrum table 237 can be created at higher speed.

In addition, when the spectrum obtaining unit 130 obtains a spectrum ofa plurality of portions of the object sample image, a spectrum table 237containing more abundant information can be created. Furthermore, thespectra stored in the spectrum table 237 are those obtained from theobject sample 11, thus they are the appropriate statistical data forcalculating an estimation operator W used for estimating the spectrum ofthe object sample 11.

FIG. 11 is a flow chart showing an estimation process of a spectrum.First, in the control unit 210, the estimation operator calculating unit255 calculates an estimation operator W indicated in the Equation (6)based on a plurality of spectra stored in the spectrum table 237 (stepS201). For that, first, the estimation operator calculating unit 255finds an average vector V based on the plurality of spectra stored inthe spectrum table 237 and, based on the average vector V, calculates anautocorrelation matrix, Rss, by the Equation (7) shown below. Here, thesuffix T denotes transposition of determinant.

R _(SS) =ΣV·V ^(T)  (7)

Thereafter, the estimation operator calculating unit 255 calculates,based on the autocorrelation matrix Rss, an estimation operator W by theEquation (6). The Equation (6) is shown again below.

W=R _(SS) H ^(t)(R _(SS) H ^(t) +R _(NN))⁻¹  (6)

Where, ( )^(t): transposed matrix, and ( )⁻¹: inverse matrix.

Thus the estimation operator W that is appropriate for estimating aspectrum of the object sample image is obtained. The obtained estimationoperator W is stored in the storage unit 230. As to the spectrum usedfor calculation of autocorrelation matrix, all of the spectra stored inthe spectrum table 237 may be used or a part thereof may be extractedfor a certain purpose by eliminating specific data. In addition, whenthe data amount is insufficient, spectra for other samples orgeneral-purpose spectra may be used together. Further, instead of anautocorrelation matrix, a covariance matrix may be used.

Thereafter, in the control unit 210, the spectrum estimating unit 257estimates a spectrum of the object sample 11 based on the pixel value ofthe pixel to be estimated, included in the virtual slide 235 (stepS203). In other words, in the spectrum estimating unit 257, theestimation value T̂(x) of the spectral transmittance at a correspondingsample point of the object sample 11 is estimated based on the pixelvalue G(x) of the pixel to be estimated by using the estimation operatorW of the aforementioned Equation (5), which is shown again below.

{circumflex over (T)}(x)=WG(x)  (5)

In this manner, spectrum estimation error can be reduced by estimatingthe estimation value T̂(x) of the spectral transmittance. The estimationvalue T̂(x) of the spectral transmittance is stored in the storage unit230.

As mentioned above, according to the virtual microscope system inaccordance with the present embodiment, the spectrum table 237 and thevirtual slide 235 can be created almost simultaneously, therebyestimating a spectrum at high speed. In addition, when the optical pathsetting unit 150 is constituted by the beam splitter 15 b, a spectrumcan be estimated at higher speed. Further, since the estimation operatorappropriate for the object sample can be calculated, estimation error inspectrum estimation can be reduced. Moreover, when the spectrumobtaining unit 130 obtains a spectrum of a plurality of portions of theobject sample image, estimation error in spectrum estimation can befurther reduced.

Second Embodiment

FIGS. 12( a) and 12(b) are diagrams for illustrating the principles of avirtual microscope system in accordance with a second embodiment of thepresent invention. In the virtual microscope system in accordance withthe present embodiment, in addition to the operation principles of thefirst embodiment described by FIGS. 2( a) and 2(b), when the spectrum 24obtained from the object sample image is registered in the spectrumtable 25, the pixel value of the pixel and the position information ofthe portion 23 where the spectrum 24 is measured are obtained from theobject sample image 21 and are stored as a data set 26 in the spectrumtable 25, thereby creating a spectrum table containing a pixel valuealmost simultaneously with creation of a virtual slide. Other than thatare the same as the principles shown in FIGS. 2( a) and 2(b).

FIG. 13 is a functional block diagram showing a configuration of thevirtual microscope system in accordance with the present embodimentbased on the aforementioned principles. In the virtual microscopesystem, the calculating unit 250 of the host system 200 has aconfiguration that is different from that shown in FIG. 5. In otherwords, the calculating unit 250 includes, in addition to the estimationoperator calculating unit 255, the spectrum estimating unit 257 and thevirtual slide creating unit 259 shown in FIG. 5, a spectrum obtainingarea pixel value calculating unit 251 for calculating a pixel value in aspectrum obtaining area from the object sample image and a spectrumselecting unit 253 for selecting a spectrum from the spectrum table 237.Other than that, the configuration is the same as that shown in FIG. 5,thus the components having the same function are assigned with the samenumerical symbols and descriptions thereof are omitted.

Operation of the main parts of the virtual microscope system inaccordance with the present embodiment is described below.

FIG. 14 is a flow chart showing a creation process of a virtual slideand a spectrum table. First, the control unit 210 performs steps S101 toS111, as in the case of the first embodiment shown in FIG. 10.Thereafter, the spectrum obtaining area pixel value calculating unit 251of the control unit 210 calculates the pixel value in the spectrumobtaining area from the corresponding object sample image (step S112 a).In this case, with respect to the image obtaining unit 110 and thespectrum obtaining unit 130, their optical axes are aligned by theoptical path setting unit 150, thereby enabling the spectrum obtainingarea pixel value calculating unit 251 to obtain the pixel in the area inwhich the spectrum is obtained.

In general, the spatial resolution of the spectrum obtaining unit 130 islower than that of the image obtaining unit 110. Therefore, the spectrumobtaining area pixel value calculating unit 251 executes convolution ofthe pixel value G of the pixel in the area in which the spectrum isobtained and the light receiving characteristic A of the spectrumobtaining unit 130 by the Equation (8), thereby calculating the pixelvalue corresponding to the spectrum based on the pixel value of thepixel in the area in which the spectrum is obtained.

$\begin{matrix}{{\hat{G}\left( {x,y} \right)} = {\sum\limits_{j = {y - D}}^{y + D}{\sum\limits_{i = {x - D}}^{x + D}{{G\left( {i,j} \right)}{A\left( {i,j} \right)}}}}} & (8)\end{matrix}$

The pixel value corresponding to the spectrum is not limited to theaforementioned value, and can be a pixel value of the central pixel inthe area in which the spectrum is obtained or the statistical valuessuch as average value, mode value, median value of the pixel values ofthe pixels in the area in which the spectrum is obtained. Further, thepixel value can be an obtained pixel value, a pixel value converted tothe color space such as L*a*b* space, or a feature value calculated froma pixel value.

The spectrum obtained at the step S111 and the pixel value calculated atthe step S112 a are stored with the information of the position at whichthe spectrum is obtained in the spectrum table 237 as a data set (stepS112 b). The following steps from S115 to S117 are the same as those ofthe first embodiment.

In this manner, as shown in FIG. 15, for example, the spectrum table 237in which the pixel values, the spectrum values and the pixel positionsare correlated as a data set can be obtained. Thus, the statistical datawhich is more appropriate for calculating the estimation operator W forestimating a spectrum of an object sample can be obtained.

FIG. 16 is a flow chart showing an estimation process of a spectrum.First, in the control unit 210, the spectrum selecting unit 253 selectsa spectrum from the spectrum table 237 based on the pixel value of thepixel to be estimated included in the virtual slide 235 (step S202 a).Thus, the spectrum selecting unit 253 first calculates the pixel valueof the pixel to be estimated and the similarity d of pixel values ofeach data set on the spectrum table 237.

For the similarity d, the statistical value such as the Euclideandistance between two pixel values, for example, is used. In this case,the Euclidean distance between the two pixel values that have beenconverted to L*a*b* space means color difference. Further, thesimilarity d may be calculated per band so that all bands satisfy theconditions or a part of the bands satisfies the conditions. Moreover,the data set on the spectrum table 237 to be compared may be limited tothe data set in which the difference between the two pixel values iswithin the arbitrary threshold. Thus search at high speed can berealized.

When the similarity d is the Euclidean distance between the two pixelvalues, the Euclidean distance between the same values is zero.Therefore, the value approaches zero as the similarity increases. Thusthe predetermined numbers of data sets are selected in order from thesmall similarity. In this case, the predetermined numbers may bedetermined from experience. In addition, the data set having thesimilarity that is smaller than the predetermined threshold may beselected. In this case, the predetermined threshold may be determinedfrom experience. Thus the statistical data similar to the pixel value ofthe pixel to be estimated can be selected. In this manner, thestatistical data most suitable for calculating the estimation operator Wfor estimating the spectrum of the pixel to be estimated can beselected. The selected data set and the calculated similarity d arestored in the storage unit 230.

Thereafter, the estimation operator calculating unit 255 of the controlunit 210 calculates the estimation operator W based on the selected dataset by the aforementioned Equation (6) (step S202 b). Thus, theestimation operator calculating unit 255 first finds the weightedaverage vector V′ based on the selected data set and the similarity d,by the Equation (9) shown below.

$\begin{matrix}{V^{\prime} = \frac{\sum\limits_{i = 1}^{N}{\frac{1}{d_{i}} \cdot T_{i}}}{\sum\limits_{i}\frac{1}{d_{i}}}} & (9)\end{matrix}$

Thereafter, the estimation operator calculating unit 255 calculates theautocorrelation matrix Rss based on the weighted average vector V′, bythe Equation (10) below. Here, the suffix T denotes transpose ofdeterminant (matrix).

R _(SS) =ΣV′·V′ ^(T)  (10)

Thereafter, the estimation operator calculating unit 255 calculates theestimation operator W based on the autocorrelation matrix Rss, by theaforementioned Equation (6). The Equation (6) is shown again below.

W=R _(SS) H ^(t)(R _(SS) H ^(t) +R _(NN))⁻¹  (6)

Where, ( )^(t): transposed matrix, and ( )⁻¹: inverse matrix.

In this manner, the estimation operator W that is most suitable forestimating the spectrum of the pixel to be estimated is obtained. Theobtained estimation operator W is stored in the storage unit 230.Thereafter, as in the case of the first embodiment shown in FIG. 11, thespectrum of the pixel to be estimated is estimated by using thecalculated estimation operator at the step S203, thereby reducing theestimation error of spectrum at each pixel.

Thus, according to the virtual microscope system in accordance with thepresent embodiment, the spectrum table 237 containing a data set, thatis, a spectrum, a pixel value of the portion from which the spectrum isobtained and the position information, is obtained almost simultaneouslywith creation of the virtual slide. Therefore, in addition to the effectby the first embodiment, a spectrum can be estimated more precisely.

For example, in the case where the virtual slide 235 is created byobtaining the object sample image 21 (tile) in the size shown in FIG. 17from the object sample 11 having a plurality of tissues, in a singlecapturing, and obtaining sample images of other tiles in series,proximal tiles are highly likely to contain the same tissue or tissueshaving a similar characteristic. Thus, in such case, when estimating aspectrum, it is more preferable to use, as the teacher data, the spectrathat are measured in the same tissue or in the tissues having a similarcharacteristic. According to the embodiment of the present embodiment,as mentioned above, the spectrum table 237 containing a data set of aspectrum, a pixel value of the portion from which the spectrum isobtained and the position information is obtained. Therefore, based onthe position information, the teacher data for the proximal position tothe pixel to be estimated can be used for estimating a spectrum. Thus aspectrum can be estimated from the teacher data of the same tissue or ofthe tissue which is highly likely to be the tissue having the similarcharacteristic, thereby enabling more precise estimation.

The present invention is not limited to the aforementioned embodiments,and many variations and modifications are available. For example, theoptical path setting unit 15 shown in FIGS. 1, 3 and 4 may be, as shownin FIGS. 18( a) and 18(b), constituted by a disposition switchingmechanism 15 c by which disposition of the image obtaining unit 16 andthe spectrum obtaining unit 17 are switched mechanically with respect tothe optical path of the observation optical system 14 to allow theobservation light to be incident on the image obtaining unit 16 in thestate of FIG. 18( a) and to allow the observation light to be incidenton the spectrum obtaining unit 17 in the state of FIG. 18( b). Further,the observation field of the object sample may be switched not only bymoving the stage, but also by moving the microscope objective lens or bymoving both the stage and the microscope objective lens.

In addition, in the second embodiment, the spectrum having the smallestsimilarity d may be used as a spectrum estimation value. In this case,although the spectrum estimation error is larger than the case where aspectrum is estimated, high speed processing is possible becausespectrum estimation is not performed. Further, the weight of theweighted average vector V′ may be set by the Equation (9), and inaddition, may be set appropriately. Moreover, an estimation operator maybe calculated by using a spectrum of other sample, or general spectrum,without performing such weighting. Further, an estimation operator maybe calculated by using a covariance matrix instead of an autocorrelationmatrix. In addition, a data set containing a spectrum may be a data setof a spectrum and a pixel value of the position from which the spectrumis obtained or a data set of a spectrum and the pixel position fromwhich the spectrum is obtained.

1. A virtual microscope system for capturing a stained sample toestimate a spectrum, comprising: an image obtaining unit for obtaining astained sample image of one or more bands of the stained sample; aspectrum obtaining unit for obtaining a spectrum at one or morepredetermined portions of the stained sample image; an optical pathsetting unit for setting an optical path of a light flux passed throughthe stained sample, with respect to the image obtaining unit and thespectrum obtaining unit, so that the spectrum of the stained sampleimage can be obtained by the spectrum obtaining unit each time the imageobtaining unit obtains the stained sample image; and a control unit forcontrolling, in two or more observation fields of the stained sample, torepeat obtaining the stained sample image by the image obtaining unitand obtaining the spectrum of the stained sample image by the spectrumobtaining unit so that a virtual slide and a spectrum table of thestained sample are created.
 2. The virtual microscope system accordingto claim 1, wherein the optical path setting unit has an optical pathswitching mirror for switching the optical path of the light flux sothat the light flux passed through the stained sample is selectivelyincident on the image obtaining unit or the spectrum obtaining unit. 3.The virtual microscope system according to claim 1, wherein the opticalpath setting unit has a disposition switching mechanism for selectivelyplacing the image obtaining unit or the spectrum obtaining unit on theoptical path of the light flux passed through the stained sample.
 4. Thevirtual microscope system according to claim 1, wherein the optical pathsetting unit has a beam splitter for splitting the optical path of thelight flux so that the light flux passed through the stained sample isincident on the image obtaining unit and the spectrum obtaining unitsimultaneously.
 5. The virtual microscope system according to claim 1,wherein the image obtaining unit is any one of an RGB camera, amonochrome camera, a two or more bands camera, and a multiband cameraprovided with a camera and an optical filter.
 6. The virtual microscopesystem according to claim 1, wherein the spectrum obtaining unit has anoptical magnification increasing unit for magnifying the stained sampleimage and obtains the spectrum from the stained sample image magnifiedby the optical magnification increasing unit.
 7. The virtual microscopesystem according to claim 1, further comprising: a spectrum obtainingposition pixel value calculating unit for obtaining a pixel value at theposition of the spectrum obtained by the spectrum obtaining unit fromthe stained sample image obtained by the image obtaining unit, whereinas the spectrum table, a spectrum table containing at least the spectrumand the pixel value is created.
 8. The virtual microscope systemaccording to claim 1, further comprising: an estimation operatorcalculating unit for calculating an estimation operator from thespectrum table; and a spectrum estimating unit for estimating a spectrumof a pixel included in the virtual slide by using the estimationoperator.
 9. The virtual microscope system according to claim 8, furthercomprising: a spectrum selecting unit for selecting a plurality ofspectra corresponding to the pixel value of a pixel included in thevirtual slide from the spectrum table, wherein the estimation operatorcalculating unit calculates an estimation operator for each pixel valuefrom the plurality of spectra selected by the spectrum selecting unit;and the spectrum estimating unit estimates the spectrum of the pixelthat makes up the virtual slide by using the estimation operator foreach of the pixel value.
 10. The virtual microscope system according toclaim 8, further comprising: a spectrum selecting unit for selecting aspectra corresponding to the pixel value of a pixel included in thevirtual slide from the spectrum table, wherein the spectrum selected bythe spectrum selecting unit is a spectrum estimation value.
 11. Thevirtual microscope system according to claim 7, wherein the pixel valuestored in the spectrum table is any one of an obtained pixel value, apixel value converted to a color space and a feature value calculatedfrom the pixel value.
 12. The virtual microscope system according toclaim 7, wherein the spectrum table consists of a data set containing atleast the spectrum, the pixel value and the information of the pixelposition from which the spectrum is obtained.
 13. The virtual microscopesystem according to claim 7, wherein the spectrum obtaining positionpixel value calculating unit calculates any one of a pixel value of thecenter pixel in an obtaining area of the spectrum, a statistical valueof the pixel value of the pixel in the obtaining area, and a valuecalculated by convolving the pixel value in the obtaining area with alight-receiving characteristic of the spectrum obtaining unit.